Golgi-localized exo-β1,3-galactosidases involved in cell expansion and root growth in Arabidopsis

Abstract

Plant arabinogalactan proteins (AGPs) are a diverse group of cell surface– and wall–associated glycoproteins. Functionally important AGP glycans are synthesized in the Golgi apparatus, but the relationships among their glycosylation levels, processing, and functionalities are poorly understood. Here, we report the identification and functional characterization of two Golgi-localized exo-β-1,3-galactosidases from the glycosyl hydrolase 43 (GH43) family in Arabidopsis thaliana. GH43 loss-of-function mutants exhibited root cell expansion defects in sugar-containing growth media. This root phenotype was associated with an increase in the extent of AGP cell wall association, as demonstrated by Yariv phenylglycoside dye quantification and comprehensive microarray polymer profiling of sequentially extracted cell walls. Characterization of recombinant GH43 variants revealed that the exo-β-1,3-galactosidase activity of GH43 enzymes is hindered by β-1,6 branches on β-1,3-galactans. In line with this steric hindrance, the recombinant GH43 variants did not release galactose from cell wall–extracted glycoproteins or AGP-rich gum arabic. These results indicate that the lack of exo-β-1,3-galactosidase activity alters cell wall extensibility in roots, a phenotype that could be explained by the involvement of galactosidases in AGP glycan biosynthesis.

The Spatio-Temporal Distribution of Cell Wall-Associated Glycoproteins During Wood Formation in Populus

“…Populus has two close orthologs of RAY1 with high relative expression in the phloem, cambium, expansion zone, and maturation zone ( Figure 7 ). In Arabidopsis, Glycoside Hydrolase family 43 (GH43) enzymes were recently shown to be Golgi-localized β-1,3-galactosidases involved in root cell expansion, possibly due to their activity toward AGP glycans ( Nibbering et al, 2020 ). The Populus genome contains only one gene encoding a GH43 enzyme ( Kumar et al, 2019 ).…”

Golgi-localized GALT7 and GALT8 participate in cellulose biosynthesis

“…The amplified DNA fragments were cloned into the pDONR207 plasmid via Gibson assembly and recombined into pHGY (Kubo et al, 2005). Stable transgenic galt7-1galt8-1 plants carrying either pGALT7:GALT7-YFP or pGALT8:GALT8-YFP were generated by Agrobacterium tumefaciens-mediated transformation, described in more detail by Nibbering et al (2020). Transgenic seedlings were selected by hypocotyl elongation on ½ Murashige and Skoog (MS) plates supplemented with 30 μg/mL hygromycin.…”

“…The bottom 10 cm of 10-week-old soil-grown plants was lyophilized and ground with a ball mill. The alcohol insoluble residue (AIR1) and starch (AIR2) were removed according to a protocol described by Nibbering et al (2020). Crystalline cellulose was quantified according to a methodology presented by Updegraff (1969).…”

“…The lines pGALT7:GALT7-YFP and pGALT8:GALT8-YFP co-expressing the marker lines SYP32-mCherry or SYP43-mCherry were generated by plant crossing, and imaged according to the methodology presented by Nibbering et al (2020).…”

On the Potential Function of Type II Arabinogalactan O-Glycosylation in Regulating the Fate of Plant Secretory Proteins

“…The fut4 fut6 double mutant showed a strong reduction in type II AG linked fucose, and oversensitivity to 150 mM NaCl compared to wild type controls. While the above mentioned studies show that type II AG structure depends on a series of biosynthetic reactions, recent data show that these O- glycans are also subject to remodeling in muro by glycoside hydrolases (GHs) such as exo-β-1-3-galactosidases ( Nibbering et al., 2020 ), β-L-arabinopyranosidases ( Imaizumi et al., 2017 ) and β-glucuronidase ( Eudes et al., 2008 ). As described for mutants in loci coding for type II AG specific GTs, mutations in type II AG active GH loci caused aberrant growth phenotypes.…”